Figure 4. Mortalin regulates NF-κB signaling in EC.

(A&B) HPAEC grown to confluence were treated with 1µg/ml MKT-077 (1h) followed by thrombin treatment (5 U/ml) for 1 h. Total cell lysates were prepared and immunoblotted with anti- IκBα antibody or anti-phospho serine (Ser⁵³⁶) antibody. Anti-RelA/p65 antibody was used to monitor loading. (C) HPAEC grown to confluence were treated with 1µg/ml MKT-077 (I h) followed by thrombin treatment (5 U/ml) for 1 h. Nuclear extracts (NE) were separated by SDS-PAGE and immunoblotted with anti-RelA/p65 antibody. Tata binding protein (TBP), a nuclear protein, was used as loading control for nuclear extracts. (D&E) HPAEC were transfected with control siRNA or mortalin siRNA using DharmaFect1. 24–36 hours later cells were treated with thrombin (5 U/ml) for 1 h. Total cell lysates were prepared and immunoblotted using anti- IκBα antibody, anti-phospho serine (Ser⁵³⁶) antibody. Anti-RelA/p65 antibody was used to monitor loading. Mortalin antibody was used to monitor mortalin depletion. (F) HPAEC were transfected with control siRNA or mortalin siRNA using DharmaFect1. 24–36 hours later cells were treated with thrombin (5 U/ml) for 1 h. NE were separated by SDS-PAGE and immunoblotted with anti-RelA/p65 antibody. TBP was used as loading control for nuclear extracts. (G) HPAEC were treated with 1µg/ml MKT-077 (I h) followed by thrombin treatment (5 U/ml) for 1 h. Nuclear extracts were prepared and assayed for DNA binding of RelA/p65 using Cayman’s NF-κB (RelA/p65) Transcription Factor Assay Kit as described in Materials and Methods. The data are the means ±S.E. (n= 4–6 for each condition). (H&I) HPAEC were treated with 1µg/ml MKT-077 (1h) followed by thrombin treatment (5 U/ml) for 1 h. Total cell lysates were subjected to immunoprecipitation with an antibody to mortalin/GRP75 (H) or antibody to RelA/p65 (I). The immunoprecipitates were then immunoblotted with antibody to RelA/p65 or mortalin/GRP75 and vice versa. TL; total lysate.